Project Summary/Abstract The applicants' long-term aim is to establish fiber-optics confocal microscopy as an intraoperative imaging modality in cardiac surgery, in particular in pediatric open heart surgery. A major risk of cardiac surgery is damage of the conduction system, which enables synchronized activation of atria and ventricles. Damage of the conduction system is irreversible, necessitates costly pacemaker insertion and is associated with significant morbidity. Despite individual variations in the conduction system, tissue discrimination during surgery is currently limited to the visual identification of anatomical landmarks. Our preliminary data indicate that microscopic images of cardiac tissue provide sufficient information for visual discrimination of various types of cardiac tissue. In this project, we will test the hypothesis that atrial and nodal tissue can be discriminated in an animal model using fiber-optics confocal microscopy and specialized imaging micro-probes. In the first specific aim, we will apply techniques established in our previous work to create and analyze three- dimensional (3D) reconstructions of atrial, sinoatrial node (SAN) and atrio-ventricular node (AVN) tissue from fixed rabbit hearts. We will use fluorescent labeling, conventional scanning confocal microscopy and methods of digital image processing to create these high-resolution micro-structural 3D models of tissue. In the second specific aim, we will develop and evaluate fiber-optics microprobes for characterization of cardiac tissue. This work will be carried out in collaboration with Mauna Kea Technologies, North America, which provides our research team with unique expertise to develop microprobes specialized for discrimination of cardiac tissue. The design and evaluation of microprobes will be based on tissue parameters extracted from the 3D reconstructions developed in the first specific aim. Several prototypes of microprobes will be designed, developed and evaluated. In the third specific aim, we will characterize atrial, SAN and AVN tissues applying fiber-optics confocal microscopy and the specialized microprobes (developed in the second specific aim) to living preparations of rabbit heart. Sets of two-dimensional image data will be acquired with our fiber-optics confocal microscope and characterized using image processing methods and visual inspection. All specific aims are designed towards testing our hypothesis that images from the fiber-optics confocal microscopy are sufficient for discrimination of atrial, SAN and AVN tissue.